Penicillin binding protein from Streptococcus pneumoniae

ABSTRACT

The invention provides isolated nucleic acid compounds encoding a novel high molecular weight PBP of  Streptococcus pneumoniae.  Also provided are vectors and transformed heterologous host cells for expressing the PBP and a method for identifying compounds that bind and/or inhibit the enzymatic activity of the PBP.

This is a division of application Ser. No. 08/731,716, filed Oct. 17,1996, now U.S. Pat. No. 5,789,202.

BACKGROUND OF THE INVENTION

This invention relates to recombinant DNA technology. In particular theinvention pertains to the cloning of a gene, pbp-nv, encoding a novelhigh molecular weight penicillin binding protein (PBP), PBP-Nv, fromStreptococcus pneumoniae and the use of said gene and its encodedprotein in a screen for new inhibitors of bacterial cell wallbiosynthesis.

The emergence of antibiotic resistance in common pathogenic bacterialspecies has justifiably alarmed the medical and research communities.The emergence and rapid spread of beta-lactam resistance inStreptococcus pneumoniae has been particularly problematic. Thisorganism is responsible for many respiratory tract infections, andresistance to beta-lactam drugs has been attributed to a modification ofone or more of the penicillin-binding proteins (PBPs). Furthermore,penicillin-resistant Streptococcus pneumoniae are frequently resistantto other commonly used antibiotics, such as erythromycin. Thesemulti-drug resistant (MDR) organisms are a real threat to humans,particularly children and the elderly. Increasingly, the only drug thatcan be used to treat infections with MDR organisms is vancomycin, andthere is considerable concern that the bacteria could also developresistance to vancomycin.

The PBPs are involved in bacterial cell wall synthesis. The cell wallcomprises a peptidoglycan layer which provides mechanical rigidity forthe bacterium. The peptidoglycan layer is composed of a sugar backbone(alternating residues of N-acetylglucosamine and N-acetylmuramic acid)attached to a pentapeptide (also referred to as “stem peptide”)containing D and L amino acid residues. In the formation of the maturepeptidoglycan, a lipid-linked disaccharide-pentapeptide is translocatedacross the cytoplasmic membrane, exposing the pentapeptide sidechains tothe cell surface. Transglycosylation of the sugar residues then leads topolymerization of the backbone sugar residues. Further stabilization ofthe nascent peptidoglycan occurs by a transpeptidation enzymaticreaction that crosslinks adjacent pentapeptide moieties. The highmolecular weight PBPs catalyze these final steps in peptidoglycansynthesis. Without the crosslinking step the peptidoglycan structure isseverely weakened and susceptible to degradation. Indeed, thecrosslinking step constitutes the target of action for antibioticcompounds such as penicillin and other beta-lactam drugs.

When effective as antibiotic agents, beta-lactam drugs interact withPBPs to form an acyl-enzyme intermediate. This intermediate is resistantto hydrolysis. Mechanistically, beta-lactam drugs act as irreversibleinhibitors. Resistance to beta-lactam drugs in Streptococcus pneumoniaearises through mutation events such that one or more low-affinity“mosaic” PBPs replace a wild-type PBP. The molecular basis of resistancehas in a few cases been correlated with specific mutations within a PBPgene. The discovery of new antibacterial compounds against thetranspeptidase domain of PBP-Nv or to an unexploited target (e.g. thetransglycosylase domain) would be particularly-useful againstStreptococcus pneumoniae infections.

SUMMARY OF THE INVENTION

The present invention is designed to meet the aforementioned need andprovides, inter alia, isolated nucleic acid molecules that encode anovel PBP from Streptococcus pneumoniae. The invention also providesprotein products encoded by the gene, in substantially purified form.

Having the cloned pbp-nv gene of Streptococcus pneumoniae enables theproduction of recombinant PBP-Nv protein and derivatives thereof for theimplementation of assays and screens to identify new inhibitorycompounds targeted at the peptidoglycan biosynthetic pathway.

In one embodiment the present invention relates to isolated gene pbp-nvthat encodes novel Streptococcus pneumoniae PBP, PBP-Nv, said genecomprising the nucleotide sequence identified as SEQ ID NO. 1.

In another embodiment the present invention relates to a novel proteinmolecule, PBP-Nv, wherein said protein molecule comprises the sequenceidentified as SEQ ID NO. 2.

In another embodiment, the present invention relates to a soluble formof PBP-Nv (designated PBP-NvS) wherein PBP-Nv^(S) comprises amino acidresidues 78 through 731, inclusive, of SEQ ID NO.2.

In a further embodiment the present invention relates to a ribonucleicacid molecule encoding PBP-Nv protein, said ribonucleic acid moleculecomprising the sequence identified as SEQ ID NO. 3:

In yet another embodiment, the present invention relates to arecombinant DNA vector that incorporates the Streptococcus pneumoniaepbp-nv gene in operable linkage to gene expression sequences enablingsaid PBP gene to be transcribed and translated in a host cell.

In still another embodiment the present invention relates to homologousor heterologous host cells that have been transformed or transfectedwith a vector carrying the cloned pbp-nv gene from Streptococcuspneumoniae such that said gene is expressed in the host cell.

In a still further embodiment, the present invention relates to a methodfor identifying compounds that bind the Streptococcus pneumoniae PBP-Nvprotein or fragment thereof.

DESCRIPTION OF THE DRAWING

FIGURE. Plasmid JAH142, useful for high level expression of theStreptococcus pneumoniae pbp-nv^(S) gene of the present invention in theheterologous procaryotic host cell Eschericia coli.

DEFINITIONS

“PBP” refers generically to a penicillin binding protein.

“PBP-Nv” refers to the novel high molecular weight PBP fromStreptococcus pneumoniae that is the subject of this invention and whichis specified by SEQ ID NO.2.

“PBP-Nv^(S)” refers to a soluble form of PBP-Nv wherein the membranespanning region and the cytoplasmic region of PBP-Nv have been removed,leaving amino acid residues 78 through 731 of SEQ ID NO.2.

“pbp-nv” refers to the Streptococcus pneumoniae genomic sequenceencoding PBP-Nv.

“pbp-nv^(S)” refers to a portion of pbp-nv that encodes PBP-Nv^(S)comprising nucleotide residues 232 through 2193 of SEQ ID NO. 1.

The terms “cleavage” or “restriction” of DNA refers to the catalyticcleavage of the DNA with a restriction enzyme that acts only at certainsequences in the DNA (viz. sequence-specific endonucleases). The variousrestriction enzymes used herein are commercially available and theirreaction conditions, cofactors, and other requirements are used in themanner well known to one of ordinary skill in the art. Appropriatebuffers and substrate amounts for particular restriction enzymes arespecified by the manufacturer or can readily be found in the literature.

The term “fusion protein” denotes a hybrid protein molecule not found innature comprising a translational fusion or enzymatic fusion in whichtwo or more different proteins or fragments thereof are covalentlylinked on a single polypeptide chain.

“Functional domain” refers to a region of a protein having one or moredistinct biological functions, for example, enzymatic activity,transmembrane anchoring, DNA binding, etc. A functional domain comprisesa sequence of amino acids, the length of which and the identity of aminoacid residues therein, may or may not be critical to function.

The term “plasmid” refers to an extrachromosomal genetic element. Thestarting plasmids herein are either commercially available, publiclyavailable on an unrestricted basis, or can be constructed from availableplasmids in accordance with published procedures. In addition,equivalent plasmids to those described are known in the art and will beapparent to the ordinarily skilled artisan.

“Recombinant DNA cloning vector” as used herein refers to anyautonomously replicating agent, including, but not limited to, plasmidsand phages, comprising a DNA molecule to which one or more additionalDNA segments can or have been added.

The term “recombinant DNA expression vector” as used herein refers toany recombinant DNA cloning vector, for example a plasmid or phage, inwhich a promoter and other regulatory elements are present to enabletranscription of the inserted DNA.

The term “vector” as used herein refers to a nucleic acid compound usedfor introducing exogenous DNA into host cells. A vector comprises anucleotide sequence which may encode one or more protein molecules.Plasmids, cosmids, viruses, and bacteriophages, in the natural state orwhich have undergone recombinant engineering, are examples of commonlyused vectors.

The terms “complementary” or “complementarity” as used herein refers tothe capacity of purine and pyrimidine nucleotides to associate throughhydrogen bonding in double stranded nucleic acid molecules. Thefollowing base pairs are complementary: guanine and cytosine; adenineand thymine; and adenine and uracil.

“Isolated nucleic acid compound” refers to any RNA or DNA sequence,however constructed or synthesized, which is locationally distinct fromits natural location.

A “primer” is a nucleic acid fragment which functions as an initiatingsubstrate for enzymatic or synthetic elongation of, for example, anucleic acid molecule.

The term “promoter” refers to a DNA sequence which directs transcriptionof DNA to RNA.

A “probe” as used herein is a labeled nucleic acid compound thathybridizes with another nucleic acid compound.

The term “hybridization” as used herein refers to a process in which asingle-stranded nucleic acid molecule joins with a complementary strandthrough nucleotide base pairing. “Selective hybridization” refers tohybridization under conditions of high stringency. The degree ofhybridization depends upon, for example, the degree of complementarity,the stringency of hybridization, and the length of hybridizing strands.

The term “stringency” refers to hybridization conditions. Highstringency conditions disfavor non-homologous basepairing. Lowstringency conditions have the opposite effect. Stringency may bealtered, for example, by temperature and salt concentration.

“Transglycosylation” refers to an enzymatic reaction catalyzed by a highmolecular weight PBP in which the sugar residues of lipid-linkeddisaccharide pentapeptide molecules are polymerized during the formationof the peptidoglycan structure of the bacterial cell wall.

“Transpeptidation” refers to an enzymatic reaction catalyzed by a highmolecular weight PBP in which the pentapeptide sidechains oflipid-linked disaccharide pentapeptde molecules are cross-linked duringthe formation of the peptidoglycan structure of the bacterial cell wall.

DETAILED DESCRIPTION

The pbp-nv gene (SEQ ID NO.1) of the present invention encodes a novelhigh molecular weight PBP of Streptococcus pneumoniae (SEQ ID NO. 2).The pbp-nv gene disclosed herein comprises a DNA sequence of 2193nucleotide base pairs (SEQ ID NO. 1). There are no interveningsequences. Those skilled in the art will recognize that owing to thedegeneracy of the genetic code (i.e. 64 codons which encode 20 aminoacids), numerous “silent” substitutions of nucleotide base pairs couldbe introduced into the sequence identified as SEQ ID NO. 1 withoutaltering the identity of the encoded amino acid(s) or protein product.All such substitutions are intended to be within the scope of theinvention.

The PBP-Nv protein defined by SEQ ID NO.2 comprises a membrane-boundprotein having several functional domains. At the amino terminal end,amino acid residues from about 1 through 56 of SEQ ID NO.2 define acytoplasmic domain. The middle portion of the molecule from about aminoacid residues 57 through 77 of SEQ ID NO.2, comprises a trans-membraneregion. At the carboxy terminal end of the molecule, which extends intothe extra-cellular environment, amino acid residues from about 78through 731 of SEQ ID NO.2 comprise a functional domain, wherein residesthe transpeptidation (viz. penicillin-binding) and transglycosylaseactivities.

The PBP-Nv protein may be modified by deletion of one or more functionaldomains. For example, the amino terminal domain and trans-membranedomains may be deleted without a loss of function at the carboxyterminal end (viz. binding of penicillin and transglycosylase activity).A deleted form of the PBP-Nv protein lacking the amino terminal andtransmembrane regions results in a soluble form of the protein,designated PBP-NvS.

Gene Isolation Procedures

Those skilled in the art will recogize that the gene of the presentinvention may be obtained by a plurality of applicable genetic andrecombinant DNA techniques including, for example, polymerase chainreaction (PCR) amplification, or de novo DNA synthesis. (See e.g., J.Sambrook et al. Molecular Cloning, 2d Ed. Chap. 14 (1989)).

Methods for constructing gene libraries in a suitable vector such as aplasmid or phage for propagation in procaryotic or eucaryotic cells arewell known to those skilled in the art. [See e.g. J. Sambrook et al.Supra]. Suitable cloning vectors are widely available.

Skilled artisans will recognize that the pbp-nv gene of Streptococcuspneumoniae comprising the present invention or fragment thereof could beisolated by PCR amplification of Streptococcus pneumoniae genomic DNA orcDNA using oligonucleotide primers targeted to any suitable region ofSEQ ID NO. 1. Methods for PCR amplification are widely known in the art.See e.g. PCR Protocols: A Guide to Method and Application, Ed. M. Inniset al., Academic Press (1990). The amplification reaction comprisesgenomic DNA, suitable enzymes, primers, and buffers, and is convenientlycarried out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk,Conn.). A positive result is determined by detecting anappropriately-sized DNA fragment following agarose gel electrophoresis.

Protein Production Methods

One embodiment of the present invention relates to the substantiallypurified protein or fragments thereof encoded by the gene disclosedherein (SEQ ID NO.1).

Skilled artisans will recognize that the protein of the presentinvention can be synthesized by any number of different methods. Theamino acid compounds of the invention can be made by chemical methodswell known in the art, including solid phase peptide synthesis orrecombinant methods. Both methods are described in U.S. Pat. No.4,617,149, incorporated herein by reference.

The principles of solid phase chemical synthesis of polypeptides arewell known in the art and may be found in general texts in the area.See, e.g., H. Dugas and C. Penney, Bioorganic Chemistry (1981)Springer-Verlag, New York, 54-92. For example, peptides may besynthesized by solid-phase methodology utilizing an Applied Biosystems430A peptide synthesizer (Applied Biosystems, Foster City, CA) andsynthesis cycles supplied by Applied Biosystems. Protected amino acids,such as t-butoxycarbonyl-protected amino acids, and other reagents arecommercially available from many chemical supply houses.

The protein of the present invention can also be produced by recombinantDNA methods using the cloned gene of Streptococcus pneumoniae disclosedherein. Recombinant methods are preferred if a high yield is desired.Expression of said cloned gene can be carried out in a variety ofsuitable host cells well known to those skilled in the art. In arecombinant method the pbp-nv or pbp-nvs gene is introduced into a hostcell by any suitable means, well known to those skilled in the art.While chromosomal integration of the cloned PBP gene is within the scopeof the present invention, it is preferred that the gene be cloned into asuitable extra-chromosomally maintained expression vector so that thecoding region of the gene is operably linked to a constitutive orinducible promoter.

The basic steps in the recombinant production of PBP-Nv or PBP-Nv^(S) ofthe present invention are:

a) constructing a natural, synthetic or semi-synthetic DNA encoding saidPBP protein;

b) integrating said DNA into an expression vector in a manner suitablefor expressing said PBP protein, as the natural protein product or as afusion protein;

c) transforming or otherwise introducing said vector into an appropriateeucaryotic or prokaryotic host cell forming a recombinant host cell,

d) culturing said recombinant host cell in a manner enabling expressionof said protein; and

e) recovering and substantially purifying said protein by any suitablemeans, well known to those skilled in the art.

Expressing Recombinant PBP Proteins in Procaryotic and Eucaryotic HostCells

In general, procaryotes are used for cloning DNA sequences and forconstructing the vectors of the present invention. Procaryotes may alsobe employed in the production of a novel PBP protein of the presentinvention. For example, the Escherichia coli K12 strain 294 (ATCC No.31446) is particularly useful for the prokaryotic expression of foreignproteins. Other strains of E. coli, bacilli such as Bacillus subtilis,enterobacteriaceae such as Salmonella typhimurium or Serratiamarcescans, various Pseudomonas species and other bacteria, such asStreptomyces, may also be employed as host cells in the cloning andexpression of the recombinant proteins of this invention.

Promoter sequences suitable for driving the expression of genes inprocaryotes include β-lactamase [e.g. vector pGX2907, ATCC 39344,contains a replicon and β-lactamase gene], lactose systems [Chang etal., Nature_(London), 275:615 (1978); Goeddel et al., Nature (London),281:544 (1979)], alkaline phosphatase, and the tryptophan (trp) promotersystem [vector pATH1 (ATCC 37695) which is designed to facilitateexpression of an open reading frame as a trpE fusion protein under thecontrol of the trp promoter]. Hybrid promoters such as the tac promoter(isolatable from plasmid pDR540, ATCC-37282) are also suitable. Stillother promoters, such as that from bacteriophage T7, whose nucleotidesequences are generally known, enable one of skill in the art to ligatesuch promoter sequences to DNA encoding the proteins of the instantinvention using linkers or adapters to supply any required restrictionsites. Still other promoters are useful for gene expression in S.pneumoniae, for example the ami promoter (J. P. Claverys etal.“Construction and evaluation of new drug-resistance cassettes forgene disruption mutagenesis in Streptococcus pneumoniae, using an amitest platform,” Gene (1995) 123-128) Promoters for use in bacterialsystems also will contain a Shine-Dalgarno sequence operably linked tothe DNA encoding the desired polypeptides. These examples areillustrative rather than limiting.

The proteins of this invention may be synthesized either by directexpression or as a fusion protein comprising the protein of interest asa translational fusion with another protein or peptide which may beremovable by enzymatic or chemical cleavage. It is often observed in theproduction of certain peptides in recombinant systems that expression asa fusion protein prolongs the lifespan, increases the yield of thedesired peptide, or provides a convenient means of purifying theprotein. A variety of peptidases (e.g. enterokinase and thrombin) thatcleave a polypeptide at specific sites or digest the peptides from theamino or carboxy termini (e.g. diaminopeptidase) of the peptide chainare known. Furthermore, particular chemicals (e.g. cyanogen bromide)will cleave a polypeptide chain at specific sites. The skilled artisanwill appreciate the modifications necessary to the amino acid sequence(and synthetic or semi-synthetic coding sequence if recombinant meansare employed) to incorporate site-specific internal cleavage sites. Seee.g., P. Carter, “Site Specific Proteolysis of Fusion Proteins”, Chapter13, in Protein Purification: From Molecular Mechanisms to Large ScaleProcesses, American Chemical Society, Washington, D.C. (1990).

In addition to procaryotes, a variety of eucaryotic microorganisms suchas yeast are suitable host cells. The yeast Saccharomyces cerevisiae isthe most commonly used eucaryotic microorganism. A number of otheryeasts such as Kluyveromyces lactis are also suitable. For expression inSaccharomyces, the plasmid YRp7 (ATCC-40053), for example, may be used.See, e.g., L. Stinchcomb, et al., Nature, 282:39 (1979); J. Kingsman etal., Gene, 7:141 (1979); S. Tschemper et al., Gene, 10:157 (1980).Plasmid YRp7 contains the TRP1 gene which provides a selectable markerfor use in a trp1 auxotrophic mutant.

Purification of Recombinantly-Produced PBP-Nv and PBP-Nv^(S)

An expression vector carrying cloned pbp-nv or pbp-nv^(S) ofStreptococcus pneumoniae is transformed or transfected into a suitablehost cell using standard methods. Cells that contain the vector arepropagated under conditions suitable for expression of the encoded PBP.If the gene is under the control of an inducible promoter, suitablegrowth conditions would incorporate an appropriate inducer.Recombinantly-produced PBP-Nv^(S) or PEP-Nv protein may be purified fromcellular extracts of transformed cells by any suitable means.Recombinantly-produced PEP-Nv is expected to be partially localized inthe host cell membrane. As such, PBP-Nv is recoverable from cellextracts and cell membranes by any suitable means, well known to thoseskilled in the art.

In a preferred process for protein purification the gene encoding thePEP of the present invention is modified at the 5″ end to incorporateseveral histidine residues at the amino terminal end of the encodedprotein product. The “histidine tag”³ enables a simplified proteinpurification method referred to as “immobilized metal ion affinitychromatography” (IMAC), essentially as described in U.S. Pat. No.4,569,794, which hereby is incorporated by reference. The IMAC methodenables rapid isolation of substantially pure protein starting from acrude cellular extract.

Other embodiments of the present invention comprise isolated nucleicacid sequences. As skilled artisans will recognize, owing to thedegeneracy of the genetic code the amino acid compounds of the inventioncan be encoded by a multitude of different nucleic acid sequences.Because these alternative nucleic acid sequences would encode the sameamino acid sequences, the present invention further comprises thesealternate nucleic acid sequences.

The nucleic acid sequences encoding SEQ ID NO:2, and disclosedsubregions therein may be produced using synthetic methodology. Thesynthesis of nucleic acids is well known in the art. See, e.g., E. L.Brown, R. Belagaje, M. J. Ryan, and H. G. Khorana, Methods inEnzymology, 68:109-151 (1979). The DNA segments corresponding to thepbp-nv or related gene sequence pbp-nv^(S) could be generated using aconventional DNA synthesizing apparatus, such as the Applied BiosystemsModel 380A or 380B DNA synthesizers (Applied Biosystems, Inc., 850Lincoln Center Drive, Foster City, Calif. 94404) which employphosphoramidite chemistry. Alternatively, phosphotriester chemistry maybe employed to synthesize the nucleic acids of this invention. [See,e.g., M. J. Gait, ed., Oliponucleotide Synthesis. A Practical Approach,(1984).]

In an alternative and preferred methodology, namely PCR, the DNAsequence comprising a portion or all of SEQ ID NO:1 can be generatedfrom Streptococcus pneumoniae genomic DNA using suitable oligonucleotideprimers complementary to SEQ ID NO:1 or region therein, as described inU.S. Pat. No. 4,889,818, which hereby is incorporated by reference.Suitable protocols for performing the PCR are widely known and aredisclosed in, for example, PCR Protocols: A Guide to Method andApplications, Ed. Michael A. Innis et al., Academic Press, Inc. (1990).

The ribonucleic acids of the present invention may be prepared using thepolynucleotide synthetic methods discussed supra, or they may beprepared enzymatically using RNA polymerase to transcribe a suitable DNAtemplate.

The most preferred systems for preparing the ribonucleic acids of thepresent invention employ the RNA polymerase from the bacteriophage T7 orthe bacteriophage SP6. These RNA polymerases are highly specific,requiring the insertion of bacteriophage-specific sequences at the 5′end of the template to be transcribed. See, J. Sambrook, et al., supra,at 18.82-18.84.

This invention also provides nucleic acids, RNA or DNA, that arecomplementary to SEQ ID NO:1 or SEQ ID NO:3.

The present invention also provides probes and primers useful for avariety of molecular biology techniques including, for example,hybridization screens of genomic or subgenomic libraries. A nucleic acidcompound comprising SEQ ID NO:1, SEQ ID NO:3 or a complementary sequencethereof, or a fragment thereof, and which is at least 18 base pairs inlength, and which will selectively hybridize to Streptococcus pneumoniaeDNA or mRNA encoding the PBP of the present invention, is provided.Preferably, the 18 or more nucleotide bases are DNA. These probes andprimers can be prepared by enzymatic methods well known to those skilledin the art (See e.g. Sambrook et al. supra). In a most preferredembodiment these probes and primers are synthesized using chemical meansas described above.

Another aspect of the present invention relates to recombinant DNAcloning vectors and expression vectors comprising the nucleic acids ofthe present invention. Many of the vectors encompassed within thisinvention are described above. The preferred nucleic acid vectors arethose that comprise DNA. The most preferred recombinant DNA vectorscomprise the isolated DNA sequence, SEQ ID NO:1. Plasmid JAH142 is anespecially preferred DNA vector for expressing the soluble form of thePBP of this invention in E. coli.

The skilled artisan understands that choosing the most appropriatecloning vector or expression vector depends upon a number of factorsincluding the availability of restriction enzyme sites, the type of hostcell into which the vector is to be transfected or transformed, thepurpose of the transfection or transformation (e.g., stabletransformation as an extrachromosomal element, or integration into thehost chromosome), the presence or absence of readily assayable orselectable markers (e.g., antibiotic resistance and metabolic markers ofone type and another), and the number of copies of the gene to bepresent in the host cell.

Vectors suitable to carry the nucleic acids of the present inventioncomprise RNA viruses, DNA viruses, lytic bacteriophages, lysogenicbacteriophages, stable bacteriophages, plasmids, viroids, and the like.The most preferred vectors are plasmids.

When preparing an expression vector the skilled artisan understands thatthere are many variables to be considered, for example, whether to use aconstitutive or inducible promoter. Inducible promoters are preferredbecause they enable high level, regulatable expression of an operablylinked gene. The skilled artisan will recognize a number of induciblepromoters that respond to a variety of inducers, for example, carbonsource, metal ions, heat, and others. The practitioner also understandsthat the amount of nucleic acid or protein to be produced dictates, inpart, the selection of the expression system. The addition of certainnucleotide sequences is useful for directing the localization of arecombinant protein. For example, a sequence encoding a signal peptidepreceding the coding region of a gene, is useful for directing theextra-cellular export of a resulting polypeptide.

Host cells harboring the nucleic acids disclosed herein are alsoprovided by the present invention. A preferred host is E. coli that hasbeen transfected or transformed with a vector that comprises a nucleicacid of the present invention.

The present invention also provides a method for constructing arecombinant host cell capable of expressing SEQ ID NO:2, or the solubleform thereof, said method comprising transforming or otherwiseintroducing into a host cell a recombinant DNA vector that comprises anisolated DNA sequence which encodes SEQ ID NO:2 or fragment thereof. Thepreferred host cell is any strain of E. coli that can accomodate highlevel expression of an exogenously introduced gene. Preferred vectorsfor expression are those which comprise SEQ ID NO:1. An especiallypreferred expression vector for use in E. coli is pJAH142, whichcomprises nucleotide residues 232 through 2193 of SEQ ID NO:1. (SeeFigure). Transformed host cells may be cultured under conditions wellknown to skilled artisans such that a recombinant protein is expressed,thereby producing in the recombinant host cell the PBP of the instantinvention.

For the purpose of identifying or developing new antibiotic compounds itis useful to determine compounds that bind to PBPs and/or inhibit thetranspeptidase or transglycosylase activity. The instant inventionprovides a screen for identifying compounds that bind PBP-Nv and/orPBP-Nv^(S) or fragment thereof, said screen comprising the steps of:

a) preparing and substantially purifying a recombinant PBP of theinvention;

b) exposing said PBP to a test compound; and

c) monitoring, by any suitable means, the binding by said compound tosaid PBP, or inhibition of enzymatic activity of said PBP by saidcompound.

The substrate for a transglycosylase assay can be made according toart-recognized methods (See e.g. DiBerardino et al. FEBS Letters, 392,184-88 (1996). For example, the lipid precursor substrate can beprepared from Streptococcus pneumoniae membranes, or from the membranesof any other suitable bacteria, UDP-Mur-Nac-pentapeptide, andUDP-N-acetyl-[¹⁴C]glucosamine (Amersham, Buckinghamshire, UK).Transglycosylase activity is measured by the production of thepeptidoglycan polymerization product essentially by mixing the substratewith a source of PBP and monitoring the amount of [¹⁴C]-label in thepeptidoglycan.

The above-disclosed screening system could also be used to identifycompounds that inhibit the transpeptidase activity of PBP-Nv or PBP-NvS.In a preferred embodiment of this aspect of the invention compounds aretested for their ability to competitively inhibit the binding of labeledpenicillin to PBP-Nv or PBP-Nv^(S).

The screening system described above provides a means to determinecompounds that interact with the PBPs of the present invention and whichmay interfere with peptidoglycan biosynthesis. This screening method maybe adapted to automated procedures such as a PANDEX® (Baxter-DadeDiagnostics) system, allowing for efficient high-volume screening forpotential inhibitory agents.

The following examples more fully describe the present invention. Thoseskilled in the art will recognize that the particular reagents,equipment, and procedures described below are merely illustrative andare not intended to limit the present invention in any manner.

EXAMPLE 1 Construction of a DNA Vector for Expressing Streptococcuspnuemoniae pbp-nv^(S) Gene in a Heterolocous Host

Plasmid JAH142 (See Figure) is an approximately 7300 base pairexpression vector suitable for expressing a modified pbp-nv^(S) inprocaryotic host E. coli. This plasmid contains an origin of replication(Ori), an ampicillin resistance gene (Amp), useful for selecting cellsthat have incorporated the vector following a tranformation procedure,and further comprises the T7 promoter in operable linkage to the codingregion of said PBP gene. Parent plasmid pET11A (obtained from Novogen,Madison, Wis.) was linearized by digestion with endonucleases NdeI andHindIII. Linearized pET11A was ligated to a DNA fragment bearing NdeIand HindIII sticky ends, comprising a modified pbp-nv^(s) gene. Thepbp-nv^(s) gene ligated into pJAH142 was modified at the 5′ end in orderto simplify purification of the encoded protein product. For thispurpose, an oligonucleotide encoding 8 histidine residues and a factorXa cleavage site, Ile-Glu-Gly-Arg, was inserted at the 3′ end of an ATGstart codon and at the 5′ end of residue 232 of SEQ ID NO: 1. Placementof the histidine residues at the amino terminus of the encoded proteinenables the IMAC protein purification procedure (See Example 4). Anadditional modification of the pbp-nvS gene ligated into pJAH142comprises two silent base pair substitutions, one at nucleotide residue270 of SEQ ID NO.1, at which a “G” replaces an “A”, and the other atnucleotide residue 777 of SEQ ID NO.1, at which a “C” replaces a “T.”These substitutions do not alter the encoded amino acid sequence.

EXAMPLE 2 Construction of a DNA Vector for Expression of pbp-nv in aheteroloaous host

The plasmid construction method outlined in Example 1 is followed toconstruct a vector for expressing PBP-Nv in a heterologous host such asE. coli. A “his-tag” oligonucleotide comprising the same structure andsequence disclosed in Example 1 is inserted at the 3′ end of the ATGinitiation codon at nucleotide position 3 of SEQ ID NO.1.

EXAMPLE 3 Expression of Streptococcus pneumoniae pbp-nvS Gene inEcherichia coli

Expression plasmid JAH142 was transformed into E. coli BL21 (DE3)(F⁻ompT[lon]hsdS r_(B) ⁻ m_(B) ⁻) using standard methods (See e.g.Sambrook et al. Supra). Transformants, chosen at random were tested forthe presence of pJAH142 by agarose gel electrophoresis using quickplasmid preparations. Id. Transformants were grown overnight at 37° C.in LB medium supplemented with 100 μg/ml ampicillin. The overnightculture was diluted into fresh LB medium and allowed to grow to anO.D.₆₀₀ of 0.4 to 0.6. At that point, expression of the vector-boundpbp-nvS gene was induced by adding 0.4 mM IPTG for a period of 3 hours.The induced-culture was then pelleted by centrifugation in preparationfor protein purification (See Example 4).

EXAMPLE 4 Purification of PBP-Nv^(S)

The recombinant cell pellet, isolated as described in the last step ofExample 3, was resuspended in 60 ml of 20 mM potassium phosphate, pH7.5. The cells were disrupted by passage through a French press,producing a cell extract that was centrifuged at 150,000×g for 1 hour.The supernatant was loaded onto a 200 ml bed-volume XK50 source-Q column(Pharmacia) equilibrated in 20 mM potassium phosphate, pH 7.5 (bufferA). The column was washed with 500 ml buffer A and eluted with a lineargradient of 0 to 1M KCl in buffer A. Fractions were pooled, tested forpenicillin binding activity, and the fractions showing binding activitywere dialyzed overnight at 4° C. against 20 mM potassium phosphate, pH 7(buffer B). The dialyzed protein sample was applied to a XK26hydroxyapatite column (60 ml bed volume) equilibrated in buffer B. Afterwashing the column with buffer B, samples were eluted with a lineargradient of 20 mM to 700 mM potassium phosphate, pH 7. Fractionsexhibiting penicillin binding activity were pooled and dialzyed against20 mM Tris, pH 8.

The PBP-Nv^(S) protein contained in the pooled fractions was purifiedfurther by immobilized metal ion affinity chromatography (IMAC),essentially as described in U.S. Pat. No. 4,569,794, the entire contentsof which is hereby incorporated by reference. Briefly, the IMACprocedure involved adding to the protein sample the following componentsat the indicated final concentrations: 0.5M NaCl, 5 mM imidazole. Thesample was loaded onto a Chelating Sepharose Fast Flow column(Pharmacia, 10 ml bed volume) and the column washed twice with 35 mleach of 20 mM Tris, pH 8, 0.5 M NaCl and 5 mM imidazole; 20 mM Tris, pH8, 0.5 M NaCl and 60 mM imidazole. The bound protein was eluted from thecolumn with 20 mM Tris, pH 8, 0.5 M NaCl, 1 M imidazole. Six fractionsof 3 ml each were collected and tested for penicillin binding activity(See Example 5).

EXAMPLE 5 Penicillin binds Streptococcus pneumoniae PBP-Nv

Protein fractions isolated from the IMAC column, as in Example 4, aretested for penicillin binding as follows. About 10 μl of the proteinsample eluted from the column is mixed with ¹²⁵I-labeled penicillin V ata final concentration of 48 μg/ml. Labeled penicillin is prepared asdescribed in Blasczak et al. “Radioiododestannylation. Convenientsynthesis of a stable penicillin derivative for rapid penicillin bindingprotein assay,” J. Labelled Compd. Radiopharm. 27, 401-06 (1989). About3 μl of a 1 μg/μl solution of sodium clavulanate is added to preventdegradation of the labeling reagent. The mixture is incubated at 35° C.for 15 minutes. Reactions are terminated by the addition of one-halfvolume SDS with boiling for 2 minutes. Aliquots of each mixture arefractionated by polyacrylamide gel electrophoresis, and radiolabeledbands are detected by exposure to X-ray film.

This method demonstrates a major labeled band at about approximately 81kilodalton (the predicted size based on amino acid sequence disclosed inSEQ ID NO.2).

EXAMPLE 6 Determination of PBP-Nv Transglycosylase activity

Radiolabelled lipid precursor for use as substrate is prepared asdescribed in H. Hara and H. Suzuki FEBS Lett. 168, 155-60 (1984).Peptidoglycan synthesis activities are determined in 50 μl reactionscontaining 50 mM PIPES, pH 6.1, 10 mM MgCl₂, 0.2 mM DTT, 1 mM ATP, 26%DMSO, PBP-Nv or PBP-Nvs sample and ¹⁴C-labelled lipid precursor. Thereaction is incubated for 30 minutes at room temperature and filteredthrough hydrophilic Durapore PVDF membranes (0.65 Jm; Millipore,Bedford, Mass.). Under these conditions the synthesized peptidoglycan isretained while the unincorporated labeled substrate is washed throughusing 0.4 M ammonium acetate in methanol. The filter bound radioactivityis determined by scintillation counting.

3 2193 base pairs nucleic acid single linear DNA (genomic) NO NO CDS1..2193 1 ATG AAA TTA GAT AAA TTA TTT GAG AAA TTT CTT TCT CTT TTT AAAAAA 48 Met Lys Leu Asp Lys Leu Phe Glu Lys Phe Leu Ser Leu Phe Lys Lys 15 10 15 GAA ACA AGT GAA CTA GAG GAC TCT GAT TCT ACT ATC TTA CGT CGC TCT96 Glu Thr Ser Glu Leu Glu Asp Ser Asp Ser Thr Ile Leu Arg Arg Ser 20 2530 CGT AGT GAT CGA AAA AAA TTA GCC CAA GTA GGT CCG ATT CGA AAA TTC 144Arg Ser Asp Arg Lys Lys Leu Ala Gln Val Gly Pro Ile Arg Lys Phe 35 40 45TGG CGT CGT TAT CAT CTA ACA AAG ATT ATC CTT ATA CTA GGT TTG AGT 192 TrpArg Arg Tyr His Leu Thr Lys Ile Ile Leu Ile Leu Gly Leu Ser 50 55 60 GCAGGC TTG CTA GTT GGA ATC TAT TTG TTT GCT GTA GCC AAG TCG ACC 240 Ala GlyLeu Leu Val Gly Ile Tyr Leu Phe Ala Val Ala Lys Ser Thr 65 70 75 80 AATGTC AAT GAT TTG CAA AAT GCC TTG AAA ACT CGG ACT CTT ATT TTT 288 Asn ValAsn Asp Leu Gln Asn Ala Leu Lys Thr Arg Thr Leu Ile Phe 85 90 95 GAC CGTGAA GAA AAA GAG GCT GGT GCC TTG TCT GGT CAA AAG GGA ACC 336 Asp Arg GluGlu Lys Glu Ala Gly Ala Leu Ser Gly Gln Lys Gly Thr 100 105 110 TAT GTTGAG CTG ACT GAC ATC AGT AAA AAC TTG CAG AAT GCT GTT ATT 384 Tyr Val GluLeu Thr Asp Ile Ser Lys Asn Leu Gln Asn Ala Val Ile 115 120 125 GCG ACAGAA GAC CGT TCT TTC TAT AAA AAT GAC GGG ATT AAC TAT GGC 432 Ala Thr GluAsp Arg Ser Phe Tyr Lys Asn Asp Gly Ile Asn Tyr Gly 130 135 140 CGT TTCTTC TTG GCT ATT GTC ACT GCT GGA CGT TCA GGT GGT GGC TCT 480 Arg Phe PheLeu Ala Ile Val Thr Ala Gly Arg Ser Gly Gly Gly Ser 145 150 155 160 ACCATT ACC CAA CAG CTG GCT AAA AAC GCC TAT TTA TCG CAG GAT CAA 528 Thr IleThr Gln Gln Leu Ala Lys Asn Ala Tyr Leu Ser Gln Asp Gln 165 170 175 ACTGTT GAG AGA AAA GCG AAA GAA TTT TTC CTT GCC TTA GAA TTA AGC 576 Thr ValGlu Arg Lys Ala Lys Glu Phe Phe Leu Ala Leu Glu Leu Ser 180 185 190 AAAAAA TAT AGT AAG GAG CAA ATT CTA ACC ATG TAC CTT AAC AAC GCT 624 Lys LysTyr Ser Lys Glu Gln Ile Leu Thr Met Tyr Leu Asn Asn Ala 195 200 205 TATTTT GGA AAT GGT GTG TGG GGT GTA GAA GAT GCG AGT AAG AAA TAC 672 Tyr PheGly Asn Gly Val Trp Gly Val Glu Asp Ala Ser Lys Lys Tyr 210 215 220 TTTGGA GTT TCT GCA TCA GAA GTG AGT CTG GAT CAA GCT GCG ACT CTG 720 Phe GlyVal Ser Ala Ser Glu Val Ser Leu Asp Gln Ala Ala Thr Leu 225 230 235 240GCA GGG ATG CTC AAG GGG CCG GAA CTG TAT AAT CCC TTG AAT TCC GTA 768 AlaGly Met Leu Lys Gly Pro Glu Leu Tyr Asn Pro Leu Asn Ser Val 245 250 255GAA GAT TCT ACT AAT CGG CGC GAT ACT GTC TTG CAG AAT ATG GTT GCA 816 GluAsp Ser Thr Asn Arg Arg Asp Thr Val Leu Gln Asn Met Val Ala 260 265 270GCA GGA TAT ATT GAT AAA AAC CAA GAA ACC GAA GCT GCT GAA GTT GAT 864 AlaGly Tyr Ile Asp Lys Asn Gln Glu Thr Glu Ala Ala Glu Val Asp 275 280 285ATG ACT TCG CAA TTG CAC GAT AAG TAT GAA GGA AAA ATC TCA GAT TAC 912 MetThr Ser Gln Leu His Asp Lys Tyr Glu Gly Lys Ile Ser Asp Tyr 290 295 300CGT TAC CCC TCT TAT TTT GAT GCG GTG GTT AAT GAA GCT GTT TCC AAG 960 ArgTyr Pro Ser Tyr Phe Asp Ala Val Val Asn Glu Ala Val Ser Lys 305 310 315320 TAT AAT CTA ACA GAG GAA GAG ATT GTC AAT AAT GGC TAC CGC ATT TAC 1008Tyr Asn Leu Thr Glu Glu Glu Ile Val Asn Asn Gly Tyr Arg Ile Tyr 325 330335 ACA GAG CTG GAC CAA AAC TAC CAA GCA AAT ATG CAG ATT GTT TAT GAA 1056Thr Glu Leu Asp Gln Asn Tyr Gln Ala Asn Met Gln Ile Val Tyr Glu 340 345350 AAC ACA TCG CTA TTT CCG AGG GCA GAG GAT GGA ACG TTT GCT CAA TCA 1104Asn Thr Ser Leu Phe Pro Arg Ala Glu Asp Gly Thr Phe Ala Gln Ser 355 360365 GGA AGT GTA GCT CTC GAA CCG AAA ACA GGG GGA GTT CGT GGA GTT GTC 1152Gly Ser Val Ala Leu Glu Pro Lys Thr Gly Gly Val Arg Gly Val Val 370 375380 GGT CAA GTT GCT GAC AAT GAT AAA ACT GGA TTC CGG AAT TTC AAC TAT 1200Gly Gln Val Ala Asp Asn Asp Lys Thr Gly Phe Arg Asn Phe Asn Tyr 385 390395 400 GCA ACC CAA TCA AAG CGT AGT CCT GGT TCT ACA ATT AAG CCT TTA GTT1248 Ala Thr Gln Ser Lys Arg Ser Pro Gly Ser Thr Ile Lys Pro Leu Val 405410 415 GTT TAT ACA CCA GCA GTT GAA GCA GGC TGG GCT TTG AAT AAG CAG TTG1296 Val Tyr Thr Pro Ala Val Glu Ala Gly Trp Ala Leu Asn Lys Gln Leu 420425 430 GAT AAC CAT ACC ATG CAG TAT GAT AGC TAT AAG GTT GAT AAC TAT GCA1344 Asp Asn His Thr Met Gln Tyr Asp Ser Tyr Lys Val Asp Asn Tyr Ala 435440 445 GGG ATC AAA ACA AGT CGA GAA GTT CCT ATG TAT CAA TCC TTG GCA GAA1392 Gly Ile Lys Thr Ser Arg Glu Val Pro Met Tyr Gln Ser Leu Ala Glu 450455 460 TCG CTT AAT CTA CCT GCT GTT GCC ACT GTT AAT GAT TTG GGT GTT GAC1440 Ser Leu Asn Leu Pro Ala Val Ala Thr Val Asn Asp Leu Gly Val Asp 465470 475 480 AAG GCT TTT GAG GCA GGC GAA AAA TTC GGA CTC AAC ATG GAA AAGGTC 1488 Lys Ala Phe Glu Ala Gly Glu Lys Phe Gly Leu Asn Met Glu Lys Val485 490 495 GAC CGT GTT CTT GGT GTC GCC TTG GGA AGC GGT GTT GAA ACC AACCCT 1536 Asp Arg Val Leu Gly Val Ala Leu Gly Ser Gly Val Glu Thr Asn Pro500 505 510 CTT CAA ATG GCT CAA GCA TAC GCT GCC TTT GCA AAT GAA GGT TTAATG 1584 Leu Gln Met Ala Gln Ala Tyr Ala Ala Phe Ala Asn Glu Gly Leu Met515 520 525 CCT GAA GCT CAT TTT ATT AGT AGA ATT GAA AAT GCT AGT GGA CAAGTT 1632 Pro Glu Ala His Phe Ile Ser Arg Ile Glu Asn Ala Ser Gly Gln Val530 535 540 ATT GCG AGT CAT AAA AAT TCA CAA AAA CGG GTG ATT GAT AAG TCTGTA 1680 Ile Ala Ser His Lys Asn Ser Gln Lys Arg Val Ile Asp Lys Ser Val545 550 555 560 GCT GAC AAG ATG ACC AGT ATG ATG TTG GGG ACT TTC ACC AACGGT ACC 1728 Ala Asp Lys Met Thr Ser Met Met Leu Gly Thr Phe Thr Asn GlyThr 565 570 575 GGT ATT AGT TCA TCG CCT GCA GAC TAT GTC ATG GCA GGG AAAACT GGA 1776 Gly Ile Ser Ser Ser Pro Ala Asp Tyr Val Met Ala Gly Lys ThrGly 580 585 590 ACA ACT GAA GCA GTT TTC AAT CCG GAG TAC ACA AGT GAC CAGTGG GTA 1824 Thr Thr Glu Ala Val Phe Asn Pro Glu Tyr Thr Ser Asp Gln TrpVal 595 600 605 ATT GGT TAT ACT CCG GAT GTA GTG ATT AGC CAC TGG CTT GGCTTT CCG 1872 Ile Gly Tyr Thr Pro Asp Val Val Ile Ser His Trp Leu Gly PhePro 610 615 620 ACC ACT GAT GAA AAT CAC TAT CTA GCT GGC TCT ACT TCA AACGGT GCA 1920 Thr Thr Asp Glu Asn His Tyr Leu Ala Gly Ser Thr Ser Asn GlyAla 625 630 635 640 GCT CAT GTC TTT AGA AAC ATT GCC AAT ACT ATT TTA CCTTAT ACG CCA 1968 Ala His Val Phe Arg Asn Ile Ala Asn Thr Ile Leu Pro TyrThr Pro 645 650 655 GGA AGT ACC TTT ACG GTT GAA AAT GCT TAT AAG CAA AATGGA ATT GCA 2016 Gly Ser Thr Phe Thr Val Glu Asn Ala Tyr Lys Gln Asn GlyIle Ala 660 665 670 CCA GCC AAT ACA AAA AGA CAA GTA CAA ACC AAT GAT AATAGC CAG ACA 2064 Pro Ala Asn Thr Lys Arg Gln Val Gln Thr Asn Asp Asn SerGln Thr 675 680 685 GAT GAT AAT TTG TCT GAT ATT CGA GGG CGT GCG CAA AGTCTA GTA GAT 2112 Asp Asp Asn Leu Ser Asp Ile Arg Gly Arg Ala Gln Ser LeuVal Asp 690 695 700 GAG GCT AGC CGG GCT ATC TCA GAT GCG AAG ATT AAG GAAAAG GCT CAA 2160 Glu Ala Ser Arg Ala Ile Ser Asp Ala Lys Ile Lys Glu LysAla Gln 705 710 715 720 ACA ATA TGG GAT TCG ATA GTC AAT CTA TTT CGC 2193Thr Ile Trp Asp Ser Ile Val Asn Leu Phe Arg 725 730 731 amino acidsamino acid linear protein 2 Met Lys Leu Asp Lys Leu Phe Glu Lys Phe LeuSer Leu Phe Lys Lys 1 5 10 15 Glu Thr Ser Glu Leu Glu Asp Ser Asp SerThr Ile Leu Arg Arg Ser 20 25 30 Arg Ser Asp Arg Lys Lys Leu Ala Gln ValGly Pro Ile Arg Lys Phe 35 40 45 Trp Arg Arg Tyr His Leu Thr Lys Ile IleLeu Ile Leu Gly Leu Ser 50 55 60 Ala Gly Leu Leu Val Gly Ile Tyr Leu PheAla Val Ala Lys Ser Thr 65 70 75 80 Asn Val Asn Asp Leu Gln Asn Ala LeuLys Thr Arg Thr Leu Ile Phe 85 90 95 Asp Arg Glu Glu Lys Glu Ala Gly AlaLeu Ser Gly Gln Lys Gly Thr 100 105 110 Tyr Val Glu Leu Thr Asp Ile SerLys Asn Leu Gln Asn Ala Val Ile 115 120 125 Ala Thr Glu Asp Arg Ser PheTyr Lys Asn Asp Gly Ile Asn Tyr Gly 130 135 140 Arg Phe Phe Leu Ala IleVal Thr Ala Gly Arg Ser Gly Gly Gly Ser 145 150 155 160 Thr Ile Thr GlnGln Leu Ala Lys Asn Ala Tyr Leu Ser Gln Asp Gln 165 170 175 Thr Val GluArg Lys Ala Lys Glu Phe Phe Leu Ala Leu Glu Leu Ser 180 185 190 Lys LysTyr Ser Lys Glu Gln Ile Leu Thr Met Tyr Leu Asn Asn Ala 195 200 205 TyrPhe Gly Asn Gly Val Trp Gly Val Glu Asp Ala Ser Lys Lys Tyr 210 215 220Phe Gly Val Ser Ala Ser Glu Val Ser Leu Asp Gln Ala Ala Thr Leu 225 230235 240 Ala Gly Met Leu Lys Gly Pro Glu Leu Tyr Asn Pro Leu Asn Ser Val245 250 255 Glu Asp Ser Thr Asn Arg Arg Asp Thr Val Leu Gln Asn Met ValAla 260 265 270 Ala Gly Tyr Ile Asp Lys Asn Gln Glu Thr Glu Ala Ala GluVal Asp 275 280 285 Met Thr Ser Gln Leu His Asp Lys Tyr Glu Gly Lys IleSer Asp Tyr 290 295 300 Arg Tyr Pro Ser Tyr Phe Asp Ala Val Val Asn GluAla Val Ser Lys 305 310 315 320 Tyr Asn Leu Thr Glu Glu Glu Ile Val AsnAsn Gly Tyr Arg Ile Tyr 325 330 335 Thr Glu Leu Asp Gln Asn Tyr Gln AlaAsn Met Gln Ile Val Tyr Glu 340 345 350 Asn Thr Ser Leu Phe Pro Arg AlaGlu Asp Gly Thr Phe Ala Gln Ser 355 360 365 Gly Ser Val Ala Leu Glu ProLys Thr Gly Gly Val Arg Gly Val Val 370 375 380 Gly Gln Val Ala Asp AsnAsp Lys Thr Gly Phe Arg Asn Phe Asn Tyr 385 390 395 400 Ala Thr Gln SerLys Arg Ser Pro Gly Ser Thr Ile Lys Pro Leu Val 405 410 415 Val Tyr ThrPro Ala Val Glu Ala Gly Trp Ala Leu Asn Lys Gln Leu 420 425 430 Asp AsnHis Thr Met Gln Tyr Asp Ser Tyr Lys Val Asp Asn Tyr Ala 435 440 445 GlyIle Lys Thr Ser Arg Glu Val Pro Met Tyr Gln Ser Leu Ala Glu 450 455 460Ser Leu Asn Leu Pro Ala Val Ala Thr Val Asn Asp Leu Gly Val Asp 465 470475 480 Lys Ala Phe Glu Ala Gly Glu Lys Phe Gly Leu Asn Met Glu Lys Val485 490 495 Asp Arg Val Leu Gly Val Ala Leu Gly Ser Gly Val Glu Thr AsnPro 500 505 510 Leu Gln Met Ala Gln Ala Tyr Ala Ala Phe Ala Asn Glu GlyLeu Met 515 520 525 Pro Glu Ala His Phe Ile Ser Arg Ile Glu Asn Ala SerGly Gln Val 530 535 540 Ile Ala Ser His Lys Asn Ser Gln Lys Arg Val IleAsp Lys Ser Val 545 550 555 560 Ala Asp Lys Met Thr Ser Met Met Leu GlyThr Phe Thr Asn Gly Thr 565 570 575 Gly Ile Ser Ser Ser Pro Ala Asp TyrVal Met Ala Gly Lys Thr Gly 580 585 590 Thr Thr Glu Ala Val Phe Asn ProGlu Tyr Thr Ser Asp Gln Trp Val 595 600 605 Ile Gly Tyr Thr Pro Asp ValVal Ile Ser His Trp Leu Gly Phe Pro 610 615 620 Thr Thr Asp Glu Asn HisTyr Leu Ala Gly Ser Thr Ser Asn Gly Ala 625 630 635 640 Ala His Val PheArg Asn Ile Ala Asn Thr Ile Leu Pro Tyr Thr Pro 645 650 655 Gly Ser ThrPhe Thr Val Glu Asn Ala Tyr Lys Gln Asn Gly Ile Ala 660 665 670 Pro AlaAsn Thr Lys Arg Gln Val Gln Thr Asn Asp Asn Ser Gln Thr 675 680 685 AspAsp Asn Leu Ser Asp Ile Arg Gly Arg Ala Gln Ser Leu Val Asp 690 695 700Glu Ala Ser Arg Ala Ile Ser Asp Ala Lys Ile Lys Glu Lys Ala Gln 705 710715 720 Thr Ile Trp Asp Ser Ile Val Asn Leu Phe Arg 725 730 2193 basepairs nucleic acid single linear mRNA NO NO 3 AUGAAAUUAG AUAAAUUAUUUGAGAAAUUU CUUUCUCUUU UUAAAAAAGA AACAAGUGAA 60 CUAGAGGACU CUGAUUCUACUAUCUUACGU CGCUCUCGUA GUGAUCGAAA AAAAUUAGCC 120 CAAGUAGGUC CGAUUCGAAAAUUCUGGCGU CGUUAUCAUC UAACAAAGAU UAUCCUUAUA 180 CUAGGUUUGA GUGCAGGCUUGCUAGUUGGA AUCUAUUUGU UUGCUGUAGC CAAGUCGACC 240 AAUGUCAAUG AUUUGCAAAAUGCCUUGAAA ACUCGGACUC UUAUUUUUGA CCGUGAAGAA 300 AAAGAGGCUG GUGCCUUGUCUGGUCAAAAG GGAACCUAUG UUGAGCUGAC UGACAUCAGU 360 AAAAACUUGC AGAAUGCUGUUAUUGCGACA GAAGACCGUU CUUUCUAUAA AAAUGACGGG 420 AUUAACUAUG GCCGUUUCUUCUUGGCUAUU GUCACUGCUG GACGUUCAGG UGGUGGCUCU 480 ACCAUUACCC AACAGCUGGCUAAAAACGCC UAUUUAUCGC AGGAUCAAAC UGUUGAGAGA 540 AAAGCGAAAG AAUUUUUCCUUGCCUUAGAA UUAAGCAAAA AAUAUAGUAA GGAGCAAAUU 600 CUAACCAUGU ACCUUAACAACGCUUAUUUU GGAAAUGGUG UGUGGGGUGU AGAAGAUGCG 660 AGUAAGAAAU ACUUUGGAGUUUCUGCAUCA GAAGUGAGUC UGGAUCAAGC UGCGACUCUG 720 GCAGGGAUGC UCAAGGGGCCGGAACUGUAU AAUCCCUUGA AUUCCGUAGA AGAUUCUACU 780 AAUCGGCGCG AUACUGUCUUGCAGAAUAUG GUUGCAGCAG GAUAUAUUGA UAAAAACCAA 840 GAAACCGAAG CUGCUGAAGUUGAUAUGACU UCGCAAUUGC ACGAUAAGUA UGAAGGAAAA 900 AUCUCAGAUU ACCGUUACCCCUCUUAUUUU GAUGCGGUGG UUAAUGAAGC UGUUUCCAAG 960 UAUAAUCUAA CAGAGGAAGAGAUUGUCAAU AAUGGCUACC GCAUUUACAC AGAGCUGGAC 1020 CAAAACUACC AAGCAAAUAUGCAGAUUGUU UAUGAAAACA CAUCGCUAUU UCCGAGGGCA 1080 GAGGAUGGAA CGUUUGCUCAAUCAGGAAGU GUAGCUCUCG AACCGAAAAC AGGGGGAGUU 1140 CGUGGAGUUG UCGGUCAAGUUGCUGACAAU GAUAAAACUG GAUUCCGGAA UUUCAACUAU 1200 GCAACCCAAU CAAAGCGUAGUCCUGGUUCU ACAAUUAAGC CUUUAGUUGU UUAUACACCA 1260 GCAGUUGAAG CAGGCUGGGCUUUGAAUAAG CAGUUGGAUA ACCAUACCAU GCAGUAUGAU 1320 AGCUAUAAGG UUGAUAACUAUGCAGGGAUC AAAACAAGUC GAGAAGUUCC UAUGUAUCAA 1380 UCCUUGGCAG AAUCGCUUAAUCUACCUGCU GUUGCCACUG UUAAUGAUUU GGGUGUUGAC 1440 AAGGCUUUUG AGGCAGGCGAAAAAUUCGGA CUCAACAUGG AAAAGGUCGA CCGUGUUCUU 1500 GGUGUCGCCU UGGGAAGCGGUGUUGAAACC AACCCUCUUC AAAUGGCUCA AGCAUACGCU 1560 GCCUUUGCAA AUGAAGGUUUAAUGCCUGAA GCUCAUUUUA UUAGUAGAAU UGAAAAUGCU 1620 AGUGGACAAG UUAUUGCGAGUCAUAAAAAU UCACAAAAAC GGGUGAUUGA UAAGUCUGUA 1680 GCUGACAAGA UGACCAGUAUGAUGUUGGGG ACUUUCACCA ACGGUACCGG UAUUAGUUCA 1740 UCGCCUGCAG ACUAUGUCAUGGCAGGGAAA ACUGGAACAA CUGAAGCAGU UUUCAAUCCG 1800 GAGUACACAA GUGACCAGUGGGUAAUUGGU UAUACUCCGG AUGUAGUGAU UAGCCACUGG 1860 CUUGGCUUUC CGACCACUGAUGAAAAUCAC UAUCUAGCUG GCUCUACUUC AAACGGUGCA 1920 GCUCAUGUCU UUAGAAACAUUGCCAAUACU AUUUUACCUU AUACGCCAGG AAGUACCUUU 1980 ACGGUUGAAA AUGCUUAUAAGCAAAAUGGA AUUGCACCAG CCAAUACAAA AAGACAAGUA 2040 CAAACCAAUG AUAAUAGCCAGACAGAUGAU AAUUUGUCUG AUAUUCGAGG GCGUGCGCAA 2100 AGUCUAGUAG AUGAGGCUAGCCGGGCUAUC UCAGAUGCGA AGAUUAAGGA AAAGGCUCAA 2160 ACAAUAUGGG AUUCGAUAGUCAAUCUAUUU CGC 2193

We claim:
 1. An isolated Penicillin Binding Protein Nv (PBP-Nv) fromStreptococcus pneumoniae having the amino acid sequence which is a SEQID NO:2.
 2. An isolated Penicillin Binding Protein Nv^(S)(PBP-Nv^(S))⁻from Streptococcus pneumoniae having the amino acidsequence which is defined by residues 78 through 731 of SEQ ID NO:2. 3.A method for identifying compounds that bind a Streptococcus pneumoniaePBP-Nv, comprising the steps of: a) admixing: i) a substantially purePBP wherein said PBP is PBP-Nv or PBP-Nv^(S); and ii) a suitable testcompound; b) measuring by any suitable means a binding by said compoundto said PBP.
 4. A method for identifying compounds that inhibit thetransglycosylase activity of a Streptococcus pneumoniae PBP-Nv,comprising the steps of: a) admixing: i) a substantially pure PBPwherein said PBP is PBP-Nv or PBP-Nv^(S); and ii) a suitable substrateand test compound; b) measuring by any suitable means inhibition of saidtransglycosylase activity.
 5. A method for identifying compounds thatinhibit the transpeptidation activity of a Streptococcus pneumoniaePBP-Nv, comprising the steps of: a) admixing: i) a substantially purePBP wherein said PBP is PBP-Nv or PBP-Nv^(S); and ii) a suitablesubstrate and test compound; b) measuring by any suitable meansinhibition of said transpeptidation activity.